Multi-sphere Cascade DNA Reaction Amplified “On-Super On–Off” PEC Sensor for Sensitive Detection of Pax-5a Gene

Pax-5a gene, as a nucleic acid biomarker closely associated with B-cell acute lymphoblastic leukemia (B-ALL), holds significant potential for early disease diagnosis. In this study, we developed a highly accurate and efficient "on-super on-off" photoelectrochemical (PEC) biosensor based on a dual-photoelectrode heterojunction system integrated with a multisphere cascade DNA amplification strategy. The designed heterojunction dual-photoelectrode platform, comprising a In₂O₃/CdS photoanode (on state) and an in situ-formed MIL-68(In)/In₂O₃ (MIO) photocathode, effectively extends the electron-hole transport pathway, enhances photogenerated charge separation, and produces high-amplitude signal output (super on state), thereby providing a robust baseline for signal transduction. Upon recognition of the target Pax-5a gene, a three-dimensional multisphere cascade DNA reaction is triggered between DNA nanospheres, generating a spatially extended DNA network structure. This structure facilitates the intercalation of doxorubicin-ferrocene carboxylic acid compound (DOX-FcCOOH), which induces a significant PEC signal quenching (off state) through efficient electron transfer, completing a distinct "on-super on-off" signal switching mechanism. This spatially confined DNA amplification strategy not only improves reaction kinetics but also enhances the anti-interference capability of the sensing system in complex biological environments. The resulting PEC biosensor demonstrates ultrasensitive detection of Pax-5a gene with a detection limit as low as 0.489 fM, along with excellent stability, reproducibility and selectivity, which offers a promising platform for early clinical diagnosis and monitoring of B-ALL.